Conjugate for mediating cell, compartment or membrane-specific transport of active substances

ABSTRACT

The present invention relates to conjugates for mediating a cell-specific, compartment-specific or membrane-specific to methods of active substances. The invention also relates to methods of preparing these conjugates as well as their use. The conjugates comprise: 
     a transport mediator for the cell membrane, 
     a cell-specific, compartment-specific or membrane-specific address protein or peptide, and 
     an active substance to be transported.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U. S. C. §371 andclaims the priority of International Patent Application No.PCT/DE00/02346 filed Jul. 14, 2000, which in turn claims priority ofGerman Patent Application No. 199 33 492.7 filed Jul. 16, 1999.

The present invention relates to conjugates for mediating cell-specific,compartment-specific or membrane-specific transport of activesubstances. The invention also relates to methods of producing saidconjugates and their use.

As is known, cellular membrane systems are largely impermeable to manysubstances (e.g. nucleic acids, proteins, chemical substances) whichshall be introduced into a cell from outside. For the introduction ofnucleic acids it is possible to penetrate cell membranes by physicalprocesses (transfection in the case of eukaryotes, transformation in thecase of prokaryotes) and biological processes (infection). In the caseof transformation, i.e. the direct take-up of the naked nucleic acid bythe cell, the cells are treated beforehand. Various methods areavailable to produce these “competent cells”. Most methods are based onthe observations made by Mandel and Higa (J. Mol. Biol. 53, pages159-163 (1970)) who were the first to show that it is possible tosubstantially increase the yields occurring when lambda-DNA is taken upby bacteria in the presence of calcium chloride. This method was usedsuccessfully for the first time by Cohen et al. (Proc. Natl. Acad. Sci.U.S.A. 69, pages 2210-2114 (1972)) for plasmid DNA and has been improvedby many modifications. Another transformation method is based on theobservation that high-frequency alternating-current fields can break upcell membranes (electroporation). This technique can be utilized toinsert naked DNA not only in prokaryotic cells but also in eukaryoticcell systems (Weaver et al., J. Cell Biochem. 51, pages 426-435 (1993)).Two very mild methods of introducing DNA into eukaryotic cells weredeveloped by Sikes et al. (Hum. Gen. Therap. 5, pages 837-840 (1994))and Yang et al. (Proc. Natl. Acad. Sci U.S.A. 87, pages 9568-9572(1990). They are based on the direct injection of the DNA into singlecells (microinjection) and on the bombardment of a cell population usingmicroprojectiles of tungsten on the surface of which the correspondingnucleic acid was bound (gene gun), respectively. In a progress parallelto the physical transformation of cells, biological infection methodshave proved their efficiency. They comprise in particular the viralintroduction of nucleic acids into cells (Chatterjee et al., Science258, pages 1485-1486 (1992); Cossett and Rusell, Gene Therapy 3, pages946-956 (1996); Bilbao et al., FASEB J. 11, pages 624-634 (1997)) andthe liposome-mediated lipofection (Bennett et al., J. Drug Targeting 5,pages 149-162 (1997)). Reference is also made to standard methods of theliposomal transport (Gao and Huang, Gene Therapy 2, pages 710-722(1995); Akhtar et al., Nucl. Acid. Res. 19, pages 5551-5559 (1991)) andpoly-L-lysine formation (Leonetti et al., Bioconj. Chem. 1(2), page 149(1990) of active substances to be able to transport them into cells.

Despite the above-listed plurality of methods of passing through thecellular membrane systems, there is no universal method serving forintroducing different active substances into cells. All of theabove-mentioned physical and biochemical methods are artificial andnon-physiological unless they make use of cell-immanent mechanisms. Itis presently not yet certain that viruses used as transport vehicles arefree of toxicity. They are often not effective and, in addition, theyare detected by the immune system.

It was therefore the object of the present invention to provide apossibility of permitting the site-directed and specific introduction ofactive substances into cells and compartments. The following demandsmust be complied with in this connection:

universal applicability

cell-specific, compartment-specific and membrane-specific introductionbehavior

high degree of effectiveness

low immunogenicity

minimization of the infection risk

sufficiently long residence time.

This object is achieved by the subject matters defined in the claims.

The inventors developed a conjugate comprising the following components:

a transport mediator for the cell membrane (“P”),

a cell-specific, compartment-specific or membrane-specific addressprotein or peptide (“AP”), and

an active substance to be transported (“W”).

The conjugate according to the invention is preferably composed asfollows:

P-AP-W

More preferably it comprises a spacer (“SP”):

P-AP-SP-W

The transport mediator for the cell membrane (abbreviated as “P” above)is a peptide or protein which can penetrate the plasma membrane. Thelength of this peptide or protein is not subject to limitation as longas it has the above property. Examples of “P” are derived preferablyfrom the penetratin family (Derossi et al., 1998, Trends Cell Biol. 8,pages 84-87) or are transportan or parts thereof (Pooga et al., TheFaseb Journal (1998), Vol. 12, page 68 et seq.), those of the penetratinfamily being preferred. An example of “P” is a penetratin having thefollowing sequence:

NH₂-RQI KIWFQNRRMKWKK-(SEQ ID NO.: 1)

(NH2-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys Trp-Lys-Lys)

Further examples of the transport protein “P” are as follows:

Viral transport protein

PTD protein transduction domain (TAT/HIV-1)

1—letter code H₂N-YGRKKRRQRRR-COOH (SEQ ID NO: 12)

3-letter code H₂N-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-

Bacterial transport molecule

TP protein transport domain TP(Eco)

1-letter code H₂N-MTRQTFWHRIKH-CQOH (SEQ ID NO: 13)

3-letter code H2N-Met-Thr-Arg-Gln-Thr-Phe-Trp-His-Arg-Ie-Lys-His

The select “P” sequence is produced biologically (purification ofnatural transport mediator proteins or cloning and expression of thesequence in a eukaryotic or prokaryotic expression system), preferablysynthetically, e.g. according to the established Merrifield method(Merrifield, J. Am. Chem. Soc. 85: 2149, 1963).

The selection of the address protein or peptide (abbreviated as “AP”above) depends on the membrane or membrane system which has to bepenetrated and the target compartment of the cell (cytoplasm, nucleus,mitochondria, chloroplast, endoplasmic reticulum) or the cell organellewhich shall be reached. The length of this address peptide or protein isnot subject to limitation as long as it comprises the property ofensuring a cell-specific, compartment-specific or membrane-specifictransport. For the introduction of active substances, in particularnucleic acids, “APs” are generally used which contain a cell-specific,compartment-specific or membrane-specific recognition signal, directingthe attached active substance to its site of action. There are the “APs”to chose from which can transport active substances in the presence orabsence of a membrane potential. The pure address sequence is usuallysufficient for a transport into the cell compartment. However, it isalso possible to chose “APs” which have a cell-specific orcompartment-specific peptidase cleavage site. In the most favorablecase, this cleavage site lies within the signal sequence but it can alsobe attached thereto by additional amino acids to ensure the cleavage ofthe address sequence after the target compartment is reached. The select“AP” sequence is produced biologically (purification of naturaltransport mediator proteins or cloning and expression of the sequence ina eukaryotic or prokaryotic expression system), preferablysynthetically, e.g. according to the established Merrifield method(Merrifield, J. Am. Chem. Soc. 85: 2149, 1963). Examples of addressproteins or peptides are as follows:

Import into the ERH₃N+-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gin-(SEQID NO: 2);

Reimport into the ER H₂N-Lys-Asp-Glu-Leu-COO⁻ (SEQ ID NO: 3);

Import into the mitochondriaH₃N+-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-(SEQID NO: 4);

Import into the nucleus—Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO: 5);

H₃N+-Pro-Lys-Lys-Lys-Arg-Lys-Val-(=nuclear localisation sequence from5V40-T antigen) (SEQ ID NO: 6);

Import into peroxisomes H₂N-Ser-Lys-Leu-COO⁻ (SEQ ID NO: 7); and

Binding to the cell membrane H₃N+-Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys (SEQID NO: 8)

Furthermore, the conjugate may optionally contain a spacer (abbreviatedas “SP” above) which is preferably located between the addressprotein/peptide and the active substance to be transported. However, itmay also be located additionally or alternatively between the transportmediator and the address protein. The spacer serves for eliminating orpositively influencing optionally existing steric interactions betweenthe components. For example, the spacer may be selected from:polylysine, polyethylene glycol (PEG), derivatives of poly-methacrylicacid or polyvinyl pyrrolidone (PVP).

A redox cleavage site, e.g. -cysteine-S-S-cysteine-O—N—H—, is preferablypresent between the transport mediator and the address protein/peptide.The binding forming between transport mediator and address protein is aredox coupling (mild cell-immanent bond by means of DMSO; Rietsch andBeckwith, 1998, Annu. Rev. Gent 32, pages 163-84):

Cysteine-SH SH-cysteine---->cystine-S-S-cystine

The active substance or active agent (abbreviated as “W” above) is notsubject to limitations. It can be chosen freely, depending on the effectwhich shall be produced in a cell. The active substance may be adiagnostic agent and/or a therapeutic agent. The conjugate may alsocomprise more than one active substance. The active substance mayoptionally be labeled, e.g. radioactively, with a dye, withbiotin/avidin, etc. The active substance may be a nucleic acid, aprotein or peptide, a chemical substance, etc. The next ones arementioned by way of example: cDNA, genomic DNA, complete genes,regulatory elements, transcription factors, molecular probes,oligonucleotides, mRNA, mTRNA, antisense RNA, antisenseoligonucleotides, plasmids, viral DNA, synthetic nucleotides, PNA(peptide nucleic acids), single amino acids and their derivatives,peptides, proteins, monoclonal and/or polyclonal antibodies,pharmaceutical active substances, chemotherapeutic agents, dyes,sensitizers, particles.

The conjugate elements “P” and “AP” are preferably synthesizedsynthetically according to the Merrifield method (Merrifield, J. Am.Chem. Soc. 85: 2149, 1963). The coupling of the other constituents (e.g.spacer and/or active substance) thereto is made by covalent chemicalbinding. The redox cleavage site is inserted chemically between “P” and“AP” by the above-mentioned redox coupling. There is also a covalentbond, preferably an acid amide bond, between an optionally presentspacer and the active substance or the address protein and the activesubstance. Possible alternatives are ether or ester bonds, depending onthe functional group(s) present in the substance to be conjugated.

The conjugate is preferably synthesized in the following steps:

1) separate peptide snythesis of “P”, “AP” and, if applicable, thespacer (e.g. according to the Merrifield method)

2) covalent bond between “AP” and active substance, if applicable, witha spacer in between,

3) redox coupling of the product from step 2) with “P” by means of redoxcoupling (e.g. in water/DMSO)

4) purification (e.g. by means of HPLC).

The conjugates according to the invention have the advantage thatirrespective of the kind and size of an active substance they canintroduce it into cells and transport it into the desired cellcompartment. Thus, an improvement of diagnostics and therapy in humanand veterinary medicines and an application in scientific research canbe anticipated. In partiuclar, the gene therapy can expect a boom onaccount of the conjugates according to the invention since completegenes including their regulatory elements become transportable. However,all of the other active substances can also be transported morespecifically to the site of action by means of the conjugates accordingto the invention, which reduces the occurrence of undesired sideeffects. It was found that conjugates up to 25 MDa can be introducedinto the cell interior. Moreover, apoptosis is often triggered, whichmight be a desired effect. The conjugates according to the inventiondistinguish themselves by a universal usability on account of theircell-specific, compartment-specific and membrane-specific introductionbehavior.

The invention is described in more detail by means of the attachedfigures:

FIG. 1 shows a conjugate according to the invention and includestransport protein RQIKIWFONRRMKWKK-(SEQ ID NO: 1) and nuclearlocalization sequence PKKKRKV (SEQ ID NO 6)

FIG. 2 shows a general diagram of the Fmoc synthesis;

FIG. 3 shows the results of the fluorescence correlation spectroscopymeasurement using AT1 cells

A) conjugate concentration: 50 nM incubation period: 5 hours

B) conjugate concentration: 5 nM incubation period: 5 hours

C) conjugate concentration: 50 nM incubation period: 24 hours

D) conjugate concentration: 5 nM incubation period: 24 hours;

FIG. 4 shows the concentration-dependent and time-dependent transport of^(rhodamine110)(L)-penetratin/RPMI medium; DU145 cells: incubation with20 μM and 100 pM final concentration;

FIG. 5 shows examples of conjugates according to the inventionincluding:

Active Transport module S-S Address Module Spacer Substance Penetratin-1S-S NLS Lys/Glyc PNA; DNA; SEQ ID NO: 1 SEQ ID NO: 6 S-ODNPTD^(TAT/HIV-1) S-S Endoplasm Retik Lys/Glyc ANTIBODY SEQ ID NO: 12 SEQID NO: 2 TP1^(AOPIEco) S-S Mitochon directed Lys/Glyc PRO-DRUGS SEQ IDNO: 13 SEQ ID NO: 4 TPF^(human) S-S Peroxis directed Lys/Glyc DRUGS SEQID NO: 8 SEQ ID NO: 7

FIG. 6 shows the production of PNA constructs, wherein the constructsinclude transport protein RQIKIWFQNRRMKWKK-(SEQ ID NO: 1) and nuclearlocalization sequence PKKKRKV (SEQ ID NO 6) wherein the active substancewas in one case a PNA having the sequence NH₂-TAC TGC GAC TCC GG-COOH(anti-sense with respect to rats P2 promoter c-myc=PNA_(AS)) (SEQ ID NO:10) and then a non-sense (random) sequence having the sequence NH₂-TTAAGG AGG CTC-COOH (=PNA_(NS)) (SEQ ID NO: 11).

FIG. 7 shows the inhibition of the proliferation of AT-1 cells byintroducing an anti-sense construct.

FIG. 8 shows the results of transport into the cytoplasm (Z) or thenucleus (N) for the conjugates produced in Example 1 for incubationperiods of 1, 3, 6, 10 and 24 hours.

The invention is described in more detail by means of the followingexamples.

EXAMPLE 1 Conjugate Comprising a Penetratin Constituent, an NLS, aPolylysine Spacer and Rhodamine

Regarding the composition of the conjugate reference is made to FIG. 1.

Penetratin: NH₂-RQIKIWFQNRRMKWKK-

NLS (nuclear localisation sequence): NH₂-PKKKRKV

Spacer (=(Lys) 2): NH—CH₂—(CH₂)₃—CHNH₂—CO—NH—CH₂— (CH₂)₃—CHNH₂—CO—NH

Penetratin sequence, NLS and spacer were synthesized separatelyaccording to the standard Fmoc method (“peptides”, H.-D. Jakubke, Chemieand Biologie Spektrum, Akad. Verl. 1996, ISBN 3-8274-0000-7). Thegeneral diagram of the Fmoc synthesis is shown in FIG. 2. Forsynthesizing the different component sequences, the first Fmoc aminoacid (purchasable from Calbiochem GmbH, D-65796 Bad Soden, Germany) isinitially attached to an insoluble polystyrene carrier resin via anacid-labile linker (=para-benzyl-oxybenzyl-alcohol-handle). Cleavage ofthe protecting group is achieved by treating the resin with 20%piperidine in dimethylformamide. The second Fmoc amino acid is linkedusing a preactivated species (e.g. succinimide, pentafluorophenylesteror p-nitrophenylester groups present in the amino acid constituents) orusing in situ activation, this was done in each case after theprotecting group was removed from the preceding amino acid by basictreatment. Each further amino acid is coupled analogously. Havingsynthesized the desired peptide, it is removed from the carrier bytreating it with 95% trifluoroacetic acid (TFA)+5% scavenger (e.g.triethylsilane), and the protecting groups are splitt off. The resultingcrude peptides are purified by preparative HPLC on a YMC ODS-A 7A S-5 μmreversed-phase column (20×250 mm) using an elution agent containing 0.1%trifluoroacetic acid in water (A) or 60% aqueous actonitrile (B). Thepeptides were eluted with a successive linear gradient from 25% B to 60%B within 40 minutes at a flow rate of 10 ml/min. The fractionscorresponding to the purified peptides were lyophilized.

The purified peptide components are treated together with 20% aqueousDMSO solution at room temperature for 5 hours, an oxidative coupling ofthe components resulting. For example, rhodamine 110 is coupled to thespacer as active substance to be transported. This is done by acid amidecoupling at the free α-amino group of the lysine spacer. The completeconjugate is then purified by means of reversed-phase HPLC.

The further conjugates according to the invention were producedanalogously:

^(AlexaTM)(L)-PTD^((TAT/HIV-1))-S-S-(L)-NLS-KK^((rhodamine110))-PNA

^(AlexaTM)(L)-TP^((IAOP/ECO))-S-S-(L)-NLS-KK^((rhodamine110))-PNA

PNA=NH₂-TTA AGG AGG CTC COOH (Example of active substance) (SEQ ID NO:11)

Alexa 350=dye (Molecular Probes, U.S.)

EXAMPLE 2 Introduction of a Conjugate According to the Invention intoCells

AT-1 (rat prostate carcinoma) and DU-145 (human prostate carcinoma, ATCCHTB-81) cells were cultured in RPMI 1640, supplementd with 10% FCS, 2 mMglutamine, 100 U/min. penicillin, 100 μg/ml streptomycin.

For fluorescence correlation spectroscopy (FCS) AT-1 or DU-145 cells aregrown on slides for 24 hours. Having changed the medium usingdyestuff-free RPMI 1640 (without phenol red), the penetratin-containingconjugate of Exmaple 1 (100 nM) is placed onto the cells using RPMI andincubated at 37° C. and with 5% CO₂ for 5, 24 or 48 hours. Thereafter,the conjugate-containing medium is removed and washed twice with 200 μlof dyestuff-free RPMI and then measured by means of FCS. Laserexcitation takes place at 488 nm and emission at 538 nm.

The conjugate is pursued on its way into the nucleus. For this, a cellis selected and focused under the light microscope. Having focused andset the laser, 100-μm steps are used for passing through the cells, andfluorescence is measured in the form of flashes by photomultipliers.Here, large molecules and small molecules migrate at differing speeds.The number of molecules diffusing in an area of 100 μm each is detected.In this way, the size of the diffused molecules can be determined bymeans of the duration of the signal. The accompanying diagram is shownin FIG. 3.

In another experiment, the kinetics by which the conjugate reaches thecytoplasm is determined by the same method. The AT-1 cells were againattached for 24 hours. The medium containing the conjugate was used asdescribed above. However, in this case, the fluorescence signal wasimmediately measured by FCS.

FCS clearly showed a strong accumultation on the cell membrane after anincubation period of 5 hours. Diffusion could not be detected. Onlyminor amounts of conjugate could be found in the cell menbrame after anincubation period of 24 hours. Attention was then attracted by anaccumulation in the nucleus which became even more intense within theobservation period of 48 hours.

For the purpose of control conjugates were used in which rhodamine 110was only bound to either penetratin or NLS. They did not show theabove-described effect of nucleus accumulation. If they succeeded at allin penetrating the cell, the conjugates were stopped at the cellmembrane of nuclear envelope where they accumulated.

As described analogously above, all of the conjugates produced inExample 1 were studied as regards their time-dependent intracellulartransport into the cytoplasm (Z) or the nucleus (N). However, differingfrom the above-mentioned incubation periods the incubation periods were1, 3, 6, 10 and 24 hours. The results are shown in Table 1.

EXAMPLE 3 Concentration-Dependent Transport

The purpose of the study was to determine to what extent theconcentration of the transport peptide^(rhodamine110)(L)-penetratin/RPMI medium influences the cellular andnucleus-directed transport in terms of time as well. A comparison wasmade between the fluorescence of 20 μM and 100 pM final concentration of^(rhodamine110)(L)-penetratin/RPMI medium. For this purpose, DU-145cells were incubated at the indicated concentrations for 1, 6, 12, 24and 48 hours. Thereafter, washing was carried out three times with RPMI(without penetratin), once with PBS and again with RPMI. Having providedthe cells with slide covers, fluorescence was determined directlyafterwards by means of CLSM (confocal laser scanning microscopy). Theresults are shown in FIG. 4. It follows therefrom that at a highconcentration of over 20 μM a non-specific transport takes place, whichsuggests cytotoxicity. However, in a lower concentration there isspecific transport into the cytoplasm.

EXAMPLE 4 Inhibition of the Proliferation of AT-1 Cells by Introductionan Anti-Sense Construct

Peptide-conjugate constructs according to FIG. 6 were produced using themethod described in Example 1 analogously. Here, the active substancewas in one case a PNA having the sequence NH₂-TAC TGC GAC TCC GG-COOH(anti-sense with respect to rats P2 promoter c-myc=PNA_(AS)) (SEQ ID NO:10)and then a non-sense (random) sequence having the nucleotide sequenceNH₂-TTA AGG AGG CTC-COOH (=PNA_(NS)) (SEQ ID NO: 11).

AT-1 cells were cultured in RPMI 1640, supplemented using 10% FCS, 2 mMglutamine, 100 U/min. penicillin, 100 μg/ml streptomycin.

AT-1 cells are grown on slides for 24 hours. Having changed the mediumusing dyestuff-free RPMI 1640 (without phenol red), the conjugates (100nM) are placed onto the cells with RPMI each and incubated at 37° C. andwith 5% CO₂ for 24, 48, 72 or 96 hours. Thereafter, theconjugate-containing medium is removed and washed twice with 200 μldyestuff-free RPMI. The cell number of AT-1 cells is determined by meansof the Coulter counting method.

Untreated AT-1 cells were used as a control. Unligated PNA_(AS)represents another control. As described analougously above, thesecontrols were incubated with the AT-1 cells.

The result of this experiment is shown in FIG. 7. The proliferation ofAT-1 was only inhibited after the administration of the anti-senseconstruct, i.e. this shows clearly that penetration of the nucleus wherethe anti-sense sequence can display the desired effect takes only placeby means of the construct according to the invention. Unligatedanti-sense sequence is as ineffective as the control or a constructwhich cannot hybridize with one of the AT-1 sequences.

13 1 16 PRT Artificial Sequence Description of the artificial sequenceTransport Mediator 1 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met LysTrp Lys Lys 1 5 10 15 2 29 PRT Artificial Sequence Description of theartificial sequence Address Peptide 2 Met Met Ser Phe Val Ser Leu LeuLeu Val Gly Ile Leu Phe Trp Ala 1 5 10 15 Thr Glu Ala Glu Gln Leu ThrLys Cys Glu Val Phe Gln 20 25 3 4 PRT Artificial Sequence Description ofthe artificial sequence Address Peptide 3 Lys Asp Glu Leu 1 4 25 PRTArtificial Sequence Description of the artificial sequence AddressPeptide 4 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala ThrArg 1 5 10 15 Thr Leu Cys Ser Ser Arg Tyr Leu Leu 20 25 5 8 PRTArtificial Sequence Description of the artificial sequence AddressPeptide 5 Pro Pro Lys Lys Lys Arg Lys Val 1 5 6 7 PRT ArtificialSequence Description of the artificial sequence Address Peptide 6 ProLys Lys Lys Arg Lys Val 1 5 7 3 PRT Artificial Sequence Description ofthe artificial sequence Address Peptide 7 Ser Lys Leu 1 8 8 PRTArtificial Sequence Description of the artificial sequence AddressPeptide 8 Gly Ser Ser Lys Ser Lys Pro Lys 1 5 9 9 PRT ArtificialSequence Description of the artificial sequence Address Peptide 9 LysLys Lys Lys Arg Lys Arg Glu Lys 1 5 10 14 DNA Artificial SequenceDescription of the artificial sequence part of a PNA 10 tactgcgact ccgg14 11 12 DNA Artificial Sequence Description of the artificial sequencepart of a PNA 11 ttaaggaggc tc 12 12 11 PRT Artificial SequenceDescription of the artificial sequence Transport Mediator 12 Tyr Gly ArgLys Lys Arg Arg Gln Arg Arg Arg 1 5 10 13 12 PRT Artificial SequenceDescription of the artificial sequence Transport Mediator 13 Met Thr ArgGln Thr Phe Trp His Arg Ile Lys His 1 5 10

What is claimed is:
 1. A conjugate for mediating a cell-specific,compartment-specific or membrane-specific transport, wherein theconjugate comprises the following components: a transport mediator forpassing through the cell membrane, a cell-specific, compartment-specificor membrane-specific address protein/peptide; and an active substance tobe transported, wherein the active substance is covalently linked to theaddress protein/peptide, and wherein a redox cleavage site is presentbetween the transport mediator and the address protein/peptide.
 2. Theconjugate according to claim 1, wherein the transport mediator can passthrough a plasma membrane.
 3. The conjugate according to claim 1,wherein the transport mediator is a member selected from the groupconsisting of: a penetratin, transportan or parts thereof, bacterialtransport protein and viral transport protein.
 4. The conjugateaccording to claim 3, wherein the penetratin has the following sequence:NH₂-RQIKIWFQNRRMKWKK-(SEQ ID NO: 1).
 5. The conjugate according to claim1, wherein the cell-specific, compartment-specific or membrane-specificaddress protein or peptide is for import into the nucleusH₃N⁺-Pro-Lys-Lys-Lys-Arg Lys-Val-(=nuclear localization sequence fromSV40-T antigen); (SEQ ID NO 6).
 6. The conjugate according to claim 1,wherein the active substance is selected from the group consisting ofnucleic acids, proteins/peptides and chemical substances.
 7. Theconjugate according to claim 1, wherein the conjugate has the followingstructure: transport mediator—address protein—active substance.
 8. Theconjugate according to claim 1, further comprising a spacer.
 9. Theconjugate according to claim 8, wherein the spacer is located betweenthe address protein and the active substance.
 10. The conjugateaccording to claim 8, wherein the spacer is a member selected from thegroup consisting of: polylysine, polyethylene glycol and polyvinylpyrrolidone.
 11. A method of preparing a conjugate comprising atransport mediator for passing through the cell membrane, acell-specific, compartment-specific or membrane-specific addressprotein/peptide; and an active substance to be transported, the methodof preparing, comprising the steps of 1) synthesizing separate peptidesof the transport mediator and address protein/peptide; 2) forming acovalent bond between the address protein/peptide and the activesubstance, and 3) redox coupling of the product from step 2) with thetransport mediator by means of redox coupling.
 12. The method accordingto claim 11, wherein the peptide synthesis is carried out according tothe Merrifield method.
 13. The method according to claim 11, wherein theredox coupling is carried out in an aqueous DMSO solution.
 14. Themethod according to claim 13, wherein a further purification stepfollows.
 15. The method according to claim 14, wherein purificationtakes place by means of HPLC.
 16. A method of transporting a desiredactive substance into a cell, a cell compartment or through a membraneof the cell, the method comprising: contacting a conjugate according toclaim 1 with a cell; and culturing the cell for a sufficient time fortransport of the conjugate into the cell, a compartment of the cell orthrough a membrane of the cell for transport of the desired activesubstance therein.
 17. A method of delivering a therapeutic agent to acell in need of such therapeutic agent, the method comprising:contacting a conjugate according to claim 3 with the cell; and culturingthe cell for a sufficient time for transport of the conjugate and thetherapeutic agent into the cell, a compartment of the cell or through amembrane of the cell.
 18. The method according to claim 11, furthercomprising: synthesizing a spacer to be covalently bonded between theaddress protein/peptide and the active substance.
 19. The conjugateaccording to claim 1, wherein the cell-specific, compartment-specificaddress protein is a nuclear localization sequence from SV40-T antigen.20. A conjugate for mediating a cell-specific, compartment-specific ormembrane-specific transport, wherein the conjugate comprises thefollowing components: a transport mediator for passing through a cellmembrane or plasma membrane, wherein the transport mediator is a memberselected from the group consisting of: a penetratin, transportan orparts thereof, bacterial transport protein and viral transport protein;a cell-specific, compartment-specific or membrane-specific addressprotein/peptide; and an active substance to be transported, wherein theactive substance is covalently linked to the address protein/peptide,and wherein a redox cleavage site is present between the transportmediator and the address protein/peptide.